Electrochemical Studies of the Effect of Temperature on the Adsorption

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Electrochemical Studies of the Effect of Temperature on the Adsorption of Yeast Alcohol Dehydrogenase at Pt Ralph K. R. Phillips, Sasha Omanovic, and Sharon G. Roscoe* Department of Chemistry, Acadia University, Wolfville, Nova Scotia, (B0P 1X0) Canada Received May 31, 2000. In Final Form: January 9, 2001 The interfacial behavior of yeast alcohol dehydrogenase (YADH) without and with the coenzyme nicotinamide adenine dinucleotide (NADH-YADH) at a Pt surface was studied over the temperature range 273-353 K in a phosphate buffer solution of pH 7.0, using cyclic voltammetry and electrochemical impedance spectroscopy. It was shown that the surface charge density and corresponding polarization resistance, resulting from protein adsorption and its oxidation, respectively, are directly proportional to the amount of adsorbed protein (surface concentration), indicating that adsorption at anodic potentials is accompanied by the transfer of charge, that is, chemisorption through carboxylate groups on the protein. The adsorption process for both proteins was described with the Langmuir adsorption isotherm, which revealed very high affinity of the proteins toward adsorption onto a Pt surface. From the calculated Gibbs energies of adsorption, it was concluded that both proteins strongly adsorb onto the Pt surface via chemisorption. The adsorption process for YADH was found to be exothermic. However, the adsorption of NADH-YADH resulted in an endothermic adsorption process as a result of the presence of the coenzyme, nicotinamide adenine dinucleotide (NAD+/NADH), in NADH-YADH which stabilizes the protein against denaturation. However, the adsorption of both proteins was found to be an entropically governed process, suggesting structural unfolding of the proteins at the electrode surface. The maximum surface concentration values indicate that there is no significant difference in the amount of adsorbed proteins between YADH and NADH-YADH in the whole temperature range investigated.

Introduction Most proteins have a strong tendency to adsorb at a solid/liquid interface. This phenomenon is of great relevance in a wide variety of both technical and natural systems. Although adsorption of proteins has been extensively studied in recent years, the process is not yet completely understood because of the intrinsic complexity of the process itself. A variety of techniques have been used to study the adsorption of proteins on solid interfaces. These include ellipsometry1-5 and radioactive labeling with 131I and 125I, but techniques such as infrared internal reflection spectroscopy (IIRS),6 differential scanning calorimetry (DSC),7 and electron microscopy8 have also been used. However, the electrochemical methods of cyclic voltammetry and electrochemical impedance spectroscopy have been used in our laboratory to develop techniques suitable for examining the interfacial behavior of proteins on stainless steel, titanium, and platinum surfaces. Proteins such as β-lactoglobulin A9 and bovine serum albumin (BSA)10 have been studied on stainless steel using electrochemical impedance spectroscopy (EIS). Fibrinogen * To whom correspondence should be addressed. Phone: (902) 585-1156. Fax: (902) 585-1114. E-mail: [email protected]. (1) Arnebrant, T.; Barton, K. J. Colloid Interface Sci. 1986, 111, 529. (2) Ivarsson, B. A.; Hegg, P.; Lundstroo¨m, K. I.; Jo¨nson, U. Colloids Surf. 1985, 13, 169. (3) Arnebrant, T.; Ivarsson, B.; Larsson, K.; Lundstro¨m, I.; Nylander, T. Prog. Colloid Polym. Sci. 1985, 70, 62. (4) Malmsten, M. J. Colloid Interface Sci. 1994, 166, 333. (5) Malmsten, M.; Lassen, B. J. Colloid Interface Sci. 1994, 166, 490. (6) Lyman, D. J.; Brash, J. L.; Chaikin, S. W.; Klein, K. G.; Carini, M. Trans.sAm. Soc. Artif. Intern. Organs 1968, 14, 250. (7) De Baillou, N.; Dejardin, P.; Schmitt, A.; Brash, J. L. J. Colloid Interface Sci. 1984, 100, 167. (8) Eberhart, R. C.; Prokop, L. D.; Wissenger, J.; Wilkov, M. A. Trans.sAm. Soc. Artif. Intern. Organs 1977, 23, 134. (9) Omanovic, S.; Roscoe, S. G. J. Colloid Interface Sci. 2000, 227, 452. (10) Omanovic, S.; Roscoe, S. G. Langmuir 1999, 15, 8315.

and BSA11 have been studied on titanium using a combination of cyclic voltammetry and EIS. A series of proteins including β-lactoglobulin A,12-14 bovine pancreatic ribonuclease A,12,14 hen egg white lysozyme,12 R-lactalbumin,15 BSA,15 bovine insulin,16 micro-peroxidase,17 and the heme proteins (cytochrome c, myoglobin, and hemoglobin)17 have been studied on platinum using cyclic voltammetry. Very little is known about the adsorption behavior of alcohol dehydrogenase (ADH). Therefore, it is of great interest to examine the adsorption behavior of this type of protein on an electrified surface. ADH catalyzes the oxidation of alcohols to aldehydes without the requirement of oxygen.18 It does however require a coenzyme, nicotinamide adenine dinucleotide (abbreviated NAD+ or NADH in its reduced form). ADH is a metalloenzyme; the active site of the enzyme contains a Zn2+ ion.19 Extensive studies have been conducted on ADH from two different sources: equine (horse) liver20-22 and yeast.23-25 (11) Jackson, D. R.; Omanovic, S.; Roscoe, S. G. Langmuir 2000, 16, 5449. (12) Roscoe, S. G.; Fuller, K. L. J. Colloid Interface Sci. 1992, 152, 429. (13) Roscoe, S. G.; Fuller, K. L.; Robitaille, G. J. Colloid Interface Sci. 1993, 160, 245. (14) Roscoe, S. G. In Modern Aspects of Electrochemistry; Bockris, J. O’M., Conway, B. E., White, R. E., Eds.; Plenum Press: New York, 1996; Vol. 29, p 319. (15) Rouhana, R.; Budge, S. M.; MacDonald, S. M.; Roscoe, S. G. Food Res. Int. 1997, 30, 303. (16) MacDonald, S. M.; Roscoe, S. G. J. Colloid Interface Sci. 1996, 184, 449. (17) Hanrahan, K. L.; MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1996, 41, 2469. (18) Dennison, M. J.; Hall, J. M.; Turner, A. P. F. Analyst 1996, 121, 1769. (19) Pietruszko, P. In Biological Electrochemistry; Dryhurst, G., Kadish, K. M., Scheller, F., Renneberg, R., Eds.; Academic Press: New York, 1982; Vol. 1, p 707. (20) Secundo, F.; Phillips, R. S. Enzyme Microb. Technol. 1996, 19, 487.

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Yeast alcohol dehydrogenase consists of four subunits. Each subunit has a molar mass of 37 500, giving a total molar mass of 150 000.26 A subunit contains an active site that binds one NAD+ and contains one Zn2+ ion.27 The zinc is both a structural and functional part of YADH and is involved in its enzymatic process.28 YADH oxidizes a variety of alcohols including propyl, isopropyl, butyl, and n-amyl alcohols. The activity of the enzyme is dependent on the number of free sulfhydryl groups.29 Upon oxidation of these groups to disulfide bonds, the activity of the enzyme diminishes. Some of the activity may return upon treatment of the enzyme with mercaptoethanol.30 Most of the studies involved with yeast alcohol dehydrogenase are those where the enzyme is immobilized on some media. As an example, lactate dehydrogenase has been studied on gold to determine its viability as a biosensor.31 An ethanol biosensor has been developed using yeast alcohol dehydrogenase and its coenzyme NAD+ immobilized by a double membrane of poly(vinyl chloride) (PVC) with a coating of cellulose triacetate (CTA). This system uses a platinum anode and a silver cathode.32 In another biosensor, a flow cell with a solution containing a mixture of YADH, NADH, and ethanol was employed to determine the concentration of alcohol in the blood.33 Studies have also been carried out on the electrochemical regeneration of NAD+.34-36 YADH is used in determining the concentration of alcohol in blood using flow injection methods in forensic and toxicology analysis.37 Yang et al.38 studied the conformational change of YADH in terms of thermal denaturation and inactivation of the enzyme. Their studies used fluorescence emission and circular dichroism spectral measurements. The present study has focused on an electrochemical investigation of the interfacial behavior of yeast alcohol dehydrogenase (YADH) in a phosphate buffer, pH 7.0, over the temperature range of 273-353 K. In addition, the effect of the presence and absence of the coenzyme, nicotinamide adenine dinucleotide, NAD+/NADH, on the adsorption behavior of YADH has been examined. The techniques of cyclic voltammetry and EIS have been used to determine the surface charge density and the components of the electrochemical equivalent circuit, respec(21) Hemmingsen, L.; Bauer, R.; Bjerrum, M. J.; Adolph, H. W.; Zeppezauer, M.; Cedergren-Zeppezauer, E. Eur. J. Biochem. 1996, 241, 546. (22) Olson, L. P.; Luo, J.; Almarsson, O ¨ .; Bruice, T. C. Biochemistry 1996, 35, 9782. (23) Wang, J.; Romero, E. G.; Reviejo, A. J. J. Electroanal. Chem. 1993, 353, 113. (24) de Marcos, S.; Galba´n, J.; Albajez, R.; Castillo, J. R. Anal. Chim. Acta 1997, 343, 117. (25) Haves, J. E.; Velick, S. F. J. Biol. Chem. 1954, 207, 225. (26) Dixon, M.; Webb, E. C. In Enzymes, 3rd ed.; Longman Group Limited: London, 1979; p 1. (27) Fersht, A. In Enzyme Structure and Mechanism, 2nd ed.; W. H. Freeman and Co.: New York, 1985; p 392. (28) Vallee, B. L.; Hoch, F. L. Proc. Natl. Acad. Sci. U.S.A. 1955, 41, 327. (29) Sumner, B.; Somers, G. F. In Chemistry and Methods of Enzymes, 3rd ed.; Academic Press Inc.: New York, 1953; p 256. (30) Buhner, M.; Sund, H. Eur. J. Biochem. 1969, 11, 73. (31) Schlereth, D. D.; KooyMan, R. P. H. J. Electroanal. Chem. 1997, 431, 285. (32) Gotoh, M.; Karube, I. Anal. Lett. 1994, 27, 273. (33) Wallace, T. C.; Leh, M. B.; Couglin, R. W. Biotechnol. Bioeng. 1975, 19, 901. (34) Jaegfeldt, H.; Torstensson, A.; Johansson, G. Anal. Chim. Acta 1978, 97, 221. (35) Couglin, R. W.; Aizawa, M.; Alexander, B. R.; Charles, M. Biotechnol. Bioeng. 1975, 17, 515. (36) Bernofsky, C.; Swan, M. Anal. Biochem. 1973, 53, 452. (37) Worsfold, P. J.; Ruzicka, J.; Hansen, E. H. Analyst 1981, 106, 1309. (38) Yang, Y.; Chen, R.; Zhou, H.-M. Biochem. Mol. Biol. Int. 1998, 45, 475.

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tively. The latter is based on computer modeling of the EIS results to fit an appropriate model of the processes occurring at the electrode surface in terms of resistance and capacitance. These values are then used to determine (i) adsorption isotherms, (ii) surface concentration of adsorbed protein, (iii) surface affinity of the protein, (iv) the Gibbs free energy of adsorption, (v) the enthalpy of adsorption, and (vi) the entropy of adsorption. These results provide information on the adsorption behavior of the proteins and the extent of denaturation on the surface. This provides a better understanding of the interfacial behavior of these proteins and their potential use in nonmediated biosensors. Experimental Section Reagents and Solutions. The stock solutions of crystallized and lyophilized baker’s yeast alcohol dehydrogenase (Sigma Chemical Co., A-3263) and crystallized and lyophilized β-NAD/ β-NADH bound baker’s yeast alcohol dehydrogenase (Sigma Chemical Co., A-7011) were prepared by dissolving reagents in 0.05 M phosphate buffer (pH 7.0). The buffer was made by dissolving monobasic KH2PO4 (Sigma Chemical Co., P-5379) in conductivity water (Nanopure, resistivity ) 18.2 MΩ cm) and adding 0.10 M sodium hydroxide (made from concentrated volumetric solution, ACP Chemical Inc.) to adjust the pH of the solution. Electrochemical Equipment. A single-compartment electrochemical cell (≈120 mL) was used in the experimental work. The working and counter electrodes were of high-purity platinum wire (99.99%, Johnson-Matthey), which was degreased by refluxing in acetone, sealed in soft glass, electrochemically cleaned by potential cycling in 0.5 M sulfuric acid, and stored in 98% sulfuric acid. The real surface area of the working electrode was obtained from the charge under the hydrogen underpotential deposition peaks when in 0.5 M sulfuric acid.39 A saturated calomel electrode (SCE) was used as the reference electrode. All potentials in this paper are referred to the SCE. Cyclic voltammetry data was obtained using a VoltaLab 40: Dynamic EIS and Voltammetry Electrochemical Laboratory (Radiometer). All measurements were recorded and graphically displayed using Voltamaster 4 (Radiometer) computer software. A Solartron Frequency Response Analyzer model 1255 and an EG&G PAR potentiostat/galvanostat Model 273, controlled by a PC using an EG&G PAR model 388 Electrochemical Impedance Software System, version 2.7, were utilized for impedance spectroscopy measurements. Impedance data were modeled using Boukamp’s ac-immittance analysis system software, Equivalent Circuit version 3.97. Experimental Methodology. All measurements were carried out in an oxygen-free solution, which was achieved by continuous purging of the cell with argon gas (Praxair). This bubbling also provided a well-mixed bulk solution. The protein solution was prepared in a separate container using phosphate buffer pH 7.0 and was allowed to equilibrate for at least 30 min in the constant-temperature bath, fitted with a Julabo P temperature regulator, at the same temperature as the electrochemical cell. After the electrode was characterized for the electrochemical technique in a phosphate buffer solution, aliquots of protein were then added to the electrochemical cell and the electrochemical measurements were repeated with each aliquot.

Results and Discussion Cyclic Voltammetry Measurements. Figure 1 shows cyclic voltammograms of a Pt electrode recorded in a phosphate buffer solution (dotted line) and in an NADHYADH-containing solution, 0.27 g L-1 in phosphate buffer (solid line). The anodic potential limit was +0.6 V, which, in a protein-free solution, resulted in a monolayer surface (39) Angerstein-Kozlowska, H. In Comprehensive Treatise of Electrochemistry; Bockris, J. O’M., Conway, B. E., Sarangapani, S., Eds.; Plenum Press: New York, 1984; Vol. 9, pp 15-59.

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Figure 1. Cyclic voltammograms of a Pt electrode in phosphate buffer, pH 7.0, recorded (dotted line) in a protein-free solution and (solid line) in a YADH-NADH-containing solution (0.27 g L-1). Scan rate, v ) 500 mV s-1; temperature, T ) 299 K.

coverage with OH species.12-16 This allows us to compare our results with those obtained by other researchers using nonelectrochemical techniques. It is evident from the presented voltammograms that addition of the protein resulted in a significant change of the voltammogram shape. Because of the adsorption of the protein at the Pt surface and corresponding blocking of OH from the surface, the charge density in the oxide region, resulting from the formation of a Pt-oxide monolayer, was suppressed by the competitive adsorption of the protein. Consequently, the oxide reduction peak in the returning scan resulted in a much smaller reduction charge. Therefore, the surface charge density, QADS, resulting from the deposition of the protein on the electrode after additions of aliquots into the bulk solution was determined from the difference between the anodic oxidation and cathodic reduction charges in the presence of the adsorbing protein, after subtracting the small difference between these two charges in the absence of protein, as follows:12-17

QADS ) [QPOo - QPOr] - [QOo - QOr]

(1)

where QOo (C cm-2) is the anodic oxidation charge density in a phosphate buffer solution, QOr is the oxide reduction charge density in a phosphate buffer solution, and QPOo is the anodic oxidation charge density and QPOr is the oxide reduction charge density in the presence of the protein. By use of eq 1, QADS was calculated from the voltammograms recorded with an anodic potential limit of +0.60 V, and the results are presented in Figure 2, which shows the dependence of the surface charge density, QADS, resulting from the adsorption of NADH-YADH onto a Pt surface, on the equilibrium concentration of the protein in the bulk solution at pH 7.0 and for a series of temperatures. At 273 K, the surface charge density initially increased with increase in concentration of NADH-YADH and then leveled off to a plateau at approximately 0.04 g L-1. This trend is observed with subsequent increases in temperature. Similar adsorption curves were obtained with YADH (not shown here). In Figure 2, the solid lines do not represent any attempt to model the data but are shown to aid visual presentation. Using surface charge density values, QADS, it is possible to calculate the protein surface concentration, Γ in mg m-2:12-17

Figure 2. Surface charge density of NADH-YADH on platinum in phosphate buffer, pH 7.0, at (b) 273, (O) 299, (9) 313, (0) 323, (4) 333, (2) 343, and ([) 353 K.

Γ)

QADSM nF

(2)

where QADS is the surface charge density in C cm-2, Mr is the molar mass of the protein in g mol-1, n is the number of electrons transferred, and F is the Faraday constant in C mol-1. Experiments performed in our laboratory, using a Pt electrode,12-17 indicated a direct involvement of carboxylate groups of proteins in the adsorption processes at the anodic Pt surface. Further, on the basis of the infrared reflection-adsorption spectroscopy and ellipsometry experiments, Liedberg et al.40 concluded that the binding of the carboxyl groups of β-lactoglobulin to a metal is through a chemical linkage (formation of an ester-type bond). Horanyi et al.41 concluded that when a carboxyl group is present the adsorption properties of a molecule are entirely determined by this group. Similar conclusions of protein interaction with surfaces through the carboxylate groups are reported in the literature.12-17,42-47 Therefore, in eq 2 the number of electrons transferred refers to the number of carboxylate groups in YADH available for binding to the Pt surface, and it has been shown9-17 that the overall reaction involves a two electron transfer process per carboxylate group. The number of carboxylate groups in YADH is 238, and the molar mass is 150 000.26 By use of the above data and eq 2, the saturated surface concentration (from the plateau) for NADH-YADH on Pt at 299 K was calculated to be 2.0 ( 0.3 mg m-2 and 1.3 ( 0.3 mg m-2 for YADH. Bowen and Gan48 investigated the adsorption of NADH-YADH at different thin layer composite microfiltration membranes and reported a maximum saturated surface concentration value of ca. 2.3 mg m-2. Using the value of the StokesEinstein radius for NADH-YADH (4.6 nm),48 it is possible (40) Liedberg, B.; Invarsson, B.; Hegg, P. O.; Lundstro¨m, I. J. Colloid Interface Sci. 1986, 114, 386. (41) Horanyi, G.; Vertes, G.; Rizmayer, E. M. J. Electroanal. Chem. 1973, 48, 207. (42) Hansen, D. C.; Luther, G. W., III; Waite, J. H. J. Colloid Interface Sci. 1994, 168, 206. (43) Fukuzaki, S.; Urano, H.; Nagata, K. J. Ferment. Bioeng. 1995, 80, 6; 1996, 81, 163. (44) Marangoni, D. G.; Smith, R. S.; Roscoe, S. G. Can. J. Chem. 1989, 67, 921. (45) Marangoni, D. G.; Wylie, I. G. N.; Roscoe, S. G. Bioelectrochem. Bioenerg. 1991, 25, 269. (46) Wylie, I. G. N.; Roscoe, S. G. Bioelectrochem. Bioenerg. 1992, 28, 367. (47) MacDonald, S. M.; Roscoe, S. G. Electrochim. Acta 1997, 42, 1189. (48) Bowen, W. R.; Gan, Q. J. Membr. Sci. 1993, 80, 165.

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Figure 3. Dependence of the maximum surface concentration on temperature derived from surface charge density QADS for the adsorption of (O) YADH and (4) NADH-YADH at platinum in phosphate buffer, pH 7.0.

to approximate the projected area of a NADH-YADH molecule lying on a smooth surface. Assuming that the Pt surface area is fully accessible to NADH-YADH molecules, the calculated quantity of NADH-YADH needed for a fully covered monolayer is ca. 3 mg m-2. Hence, the experimentally measured maximum amount of the protein adsorbed on the Pt electrode ranges from 43% (YADH) to 67% (NADH-YADH) of a full monolayer at 299 K. The results presented in Figure 2 show that with increase in temperature, the surface charge density, that is, plateau surface concentration, values increase, too. This is clearly shown in Figure 3, that represents the dependence of the plateau surface concentration values, Γmax, of YADH and NADH-YADH on the temperature. The curves in Figure 3 demonstrate that with increase in temperature from 273 to 299 K the saturated surface concentration, Γmax, of both types of protein slightly increases and at 313 K it reaches a monolayer level (3 mg m-2). However, the increase in temperature facilitates increased adsorption of protein. This may result from association or agglomeration of protein in the bulk solution at these temperatures, followed by adsorption of these associated species. Hence, at 333 K the experimental results are consistent with the formation of the protein layer two molecules thick. With further increase in temperature, the maximum surface concentration slightly decreases. The denaturation temperature for the enzyme is between 333 and 343 K,49 and therefore the drop in the plateau surface concentration noticed at 343 K may be due to the conformational changes occurring upon denaturation. However, with the further increase in temperature the maximum surface concentration increases again, thus again indicating increased adsorption of the protein. The values of the surface concentration above 343 K indicate that the thickness of the protein layer is the equivalent of three molecules. The exchange of charge through the adsorbed protein molecules is quite possible because of the existence of a relatively strong electric field at the anodic potential limit used in the experiments (+0.6 V). Our previous experiments with other proteins have also shown that the surface adsorption behavior mimics the protein behavior in the bulk solution, that is, conformational changes, the formation/dissociation of monomers, dimers, and tetramers, denaturation, and agglomeration. 12-17 (49) Nath, S.; Sathpathy, G. R.; Mantri, R.; Deep, S.; Ahluwalia, J. C.; J. Chem. Soc., Faraday Trans. 1997, 93, 3351.

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Figure 4. Langmuir adsorption isotherm of NADH-YADH adsorbed onto a Pt surface in phosphate buffer, pH 7.0, at 313 K. The symbols are experimental values, and the solid line is the best fit. Inset: Experimental data (symbols) fitted using the Langmuir isotherm equation and calculated parameters (Γmax and BADS).

According to the literature,42,33,50-52 adsorption of proteins onto solid surfaces has been described by the Langmuir isotherm:

Γ)

BADSΓmaxc 1 + BADSc

(3)

in which c (mol cm-3) is the equilibrium concentration of the adsorbate in the bulk solution, Γ (mol cm-2) is the amount of protein adsorbed, that is, surface concentration, Γmax (mol cm-2) is the maximum value of Γ (plateau or saturated surface concentration), and the parameter BADS (cm3 mol-1) reflects the affinity of the adsorbate molecules toward adsorption sites. Equation 3 can be rearranged to give

1 c c ) + Γ BADSΓmax Γmax

(4)

If the Langmuir isotherm is valid for an observed system, a plot of c/Γ versus concentration c should yield a straight line with parameters Γmax and BADS derived from the slope and intercept, respectively. As an example, adsorption of NADH-YADH is presented in Figure 4, and indeed the c/Γ versus c dependence is linear, with a correlation coefficient r2 ) 0.9999. The calculated maximum surface coverage Γmax was found to be 3.1 ( 0.1 mg m-2, which is the same as the experimentally obtained value (Figure 3). By use of the parameters obtained by the fitting procedure, the isotherm for the adsorption of NADHYADH onto the Pt surface at 313 K was calculated and plotted according to eq 3 (see inset in Figure 4). Good agreement between experimental and simulated values confirmed the applicability of the Langmuir isotherm in the description of the adsorption of both types of alcohol dehydrogenase onto the Pt surface. The applicability of the Langmuir isotherm over the whole temperature range indicates that the formation of protein layers of different thicknesses is due to temperature-dependent protein (50) Brash, J. L.; ten Hove, P. J. Biomater. Sci., Polym. Ed. 1993, 4, 591. (51) Klinger, A.; Steinberg, D.; Kohavi, D.; Sela, M. N. J. Biomed. Mater. Res. 1997, 36, 387. (52) LeDuc, C.; Ten Hove, P.; Park, S.; Vroman, L.; Brash, J.; Leonard, E. F. J. Biomater. Sci., Polym. Ed. 1995, 7, 531.

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Haynes et al.54 showed that the enthalpy of adsorption of protein is a sum of individual enthalpies related to (i) changes in the state of hydration of the sorbent surface ∆Hhyd, (ii) association/dissociation of protons with charged groups on the protein surface ∆HH+, (iii) overlap of electric fields ∆Hel, (iv) contribution to the incorporation of ions (other than protons) in the adsorbed layer ∆Hion, and (v) structural rearrangement in the protein molecule ∆Hstr.pr.. Therefore, a value of the adsorption enthalpy ∆HADS depends on the contributions of each of these individual enthalpies:54

∆HADS ) ∆Hhyd + ∆HH+ + ∆Hel + ∆Hion + ∆Hstr.pr. (6)

Figure 5. Dependence of the Gibbs free energy of adsorption on the temperature for NADH-YADH adsorbed onto platinum in phosphate buffer, pH 7.0. Table 1. Enthalpy and Entropy Values for Adsorption of YADH and NADH-YADH at a Pt Electrode protein

∆HADS/kJ mol-1

∆SADS/J mol-1 K-1

YADH NADH-YADH

-12 ( 1 17 ( 1

137 ( 7 229 ( 3

behavior in the bulk solution, such as association or agglomeration, at each temperature, followed by adsorption of the associated species. The parameter BADS, which reflects the affinity of the adsorbate molecules toward adsorption sites at a constant temperature, can be presented as53

BADS )

(

)

-∆GADS 1 exp 55.5 RT

(5)

where R (J mol-1 K-1) is the gas constant, T (K) is the temperature, ∆GADS (J mol-1) is the Gibbs free energy of adsorption, and 55.5 is the molar concentration of the water (mol dm-3), which is used as a solvent. From this equation, the Gibbs free energy of adsorption of YADH onto the Pt surface in phosphate buffer solution at 313 K was calculated to be -55 ( 3 kJ mol-1. Such a high value indicates strong adsorption of YADH onto the Pt surface via chemisorption. A chemisorption process at the electrode surface with a high affinity or equilibrium constant would dominate or mask any physisorption processes that might also be occurring. Chemisorption processes are commonly described by the Langmuir adsorption isotherm. The initial chemisorption interaction of those carboxylate groups in direct contact with the electrode surface during the protein adsorption process could also account for the observed excellent agreement of our experimental results with this isotherm. This also suggests that lateral interactions are significantly weaker than the protein/ surface interactions controlled by the chemisorption process. Figure 5 shows the dependence of the Gibbs free energy of adsorption on temperature for NADH-YADH. The negative Gibbs free energy increases linearly with temperature. From the slope and the intercept of the line, the values of entropy ∆SADS and enthalpy ∆HADS of adsorption were calculated, respectively, and are presented in Table 1 for both types of protein. (53) Gomma, G. K.; Wahdan, M. H. Mater. Chem. Phys. 1994, 39, 142.

The relatively small ∆HADS values (-12 kJ mol-1 for YADH and 17 kJ mol-1 for NADH-YADH) indicate that the enthalpies on the right-hand side of the equation mostly cancel. From the result presented here, it is not possible to determine the enthalpy value of each individual subprocess, but Haynes et al.54 showed that values of ∆Hstr.pr. are large and endothermic because of the loss of favorable intramolecular interactions within the protein when it adsorbs and unfolds on the sorbent surface (the transition enthalpy for thermal denaturation of YADH at pH 7.4 is ∆H ) 630 kJ mol-1 49) and values of ∆Hion are also large but exothermic because of the water-water hydrogen bond formation which accompanies the transfer of ions (phosphate anions in our case) from water to the adsorbed layer. The enthalpy changes associated with the remaining subprocesses are predicted to be relatively small but together could make a significant contribution to the sign and magnitude of ∆HADS.54 The enthalpy values ∆HADS presented in Table 1 show that the adsorption of YADH onto the Pt surface is an exothermic process, whereas the adsorption of NADH-YADH is an endothermic process. This observed difference in the enthalpy values (29 kJ mol-1) is probably due to the fact that the coenzyme, NAD+/ NADH, in NADH-YADH is strongly bound to the active site of the enzyme and in that way stabilizes the protein against denaturation, that is, increases its ∆Hstr.pr. value. Accordingly, the adsorption enthalpy for NADH-YADH is more positive, compared with the value obtained for YADH (Table 1). In addition, for each enzyme there are four active sites that include the coenzyme, NAD+/ NADH.55 The relatively small ∆HADS values (Table 1) indicate that the net influence of enthalpy on adsorption of NADHYADH and YADH on Pt is minor under the experimental conditions applied in our experiments. However, from the calculated thermodynamic values it is apparent that the gain in entropy actually represents the driving force for the adsorption of the proteins onto the platinum surface, because the T∆SADS product ranges from 37 to 48 kJ mol-1 for YADH and from 62 to 81 kJ mol-1 for NADH-YADH, depending on the temperature, which is considerably higher than the enthalpy values (-12 and 17 kJ mol-1, respectively). The structure changes when the protein adsorbs onto a surface. Hence, the induced structural changes can lead to a considerable entropy gain, which might be, as we see here, the driving force for adsorption. It has been shown in the literature54,56-58 that proteins with low native-state stabilities possess a strong driving force for adsorption related to breakdown of native tertiary (54) Haynes, C. A.; Sliwinsky, E.; Norde, W. J. Colloid Interface Sci. 1994, 164, 394. (55) Sund, H.; Theorell, H. In The Enzymes; Boyer, P., Lardy, H., Myrba¨ck, K., Eds.; Academic Press: New York, 1963; Vol. 7, p 25. (56) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87.

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Phillips et al.

Figure 6. Nyquist plots of a Pt electrode in phosphate buffer, pH 7.0, recorded at +0.60 V and 313 K (O) in a protein-free solution and (4) in a YADH-NADH-containing solution (0.27 g L-1). The symbols are experimentally measured values, and the lines are simulated values. Inset: Equivalent electrical circuit used to model the EIS data.

and partially secondary structure; in other words, adsorption is driven by an increase in the conformational entropy of the protein. Large positive entropy values could arise from the unfolding of protein molecules upon adsorption. It has been shown59 that the increased rotational freedom of the polypeptide backbone which results from the complete unfolding of a native protein will lead to an entropy gain of 10-100 J K-1 per mole of amino acid residue. Electrochemical Impedance Spectroscopy Measurements. The EIS technique was applied to further investigate the adsorption of both proteins at the platinum surface. To ensure complete characterization of the interface and surface processes, EIS measurements were made over seven frequency decades, from 100 kHz to 10 mHz, at a constant potential of +0.6 V. Figure 6 shows an example of EIS spectra recorded in a protein-free solution (circles) and in a solution containing 0.27 g L-1 of NADH-YADH (triangles). The presentation of the data in the form of a Nyquist impedance (complex) plot (Figure 6) clearly reveals the presence of just one time constant. Thus, a simple Randles electrical equivalent circuit (EEC) (inset in Figure 6) was introduced to model the experimental values using a nonlinear least-square fit analysis (NLLS) software.60 A very good agreement between the experimental (symbols) and simulated (lines) values was obtained. In addition, simulated spectra are extended to the very low frequencies (Figure 6, dotted lines) in order to aid visual demonstration of the closed semicircles. Instead of pure capacitance (C), a constant phase element (CPE) was introduced in the modeling procedure. This is due to a distribution of the relaxation times as a result of inhomogeneities present on the microscopic level at the electrode/electrolyte interface.61 Electronic elements in the inset in Figure 6 have the following meanings: Rel is the resistance of the electrolyte between the working and the reference electrodes, Rp is the polarization resistance, and CPEdl is the capacitance of the electric double layer at the electrode/electrolyte interface, which is represented by the constant phase element and its exponent n. (57) Van Wagenen, R. A.; Rockhold, S.; Andrade, J. D. Adv. Chem. Ser. 1982, 199, 351. (58) Bentaleb, A.; Abele, A.; Haikel, Y.; Schaaf, P.; Voegel, J. C. Langmuir 1998, 14, 6493. (59) DeWit, J. N.; Klarenbeek, G. J. Dairy Sci. 1984, 67, 2701. (60) Boukamp, B. A. In Equivalent Circuit Users Manual; Report CT88/265/128; University of Twente, Department of Chemical Technology: The Netherlands, 1989. (61) Omanovic, S.; Metikos-Hukovic, M. Thin Solid Films 1995, 266, 31.

Figure 7. Inverse of polarization resistance for NADH-YADH oxidation on platinum in phosphate buffer, pH 7.0, at 313 K. Inset: Langmuir adsorption isotherm of NADH-YADH adsorbed onto a Pt surface in phosphate buffer, pH 7.0, at 313 K. The symbols are experimental values, and the solid line is the best fit.

Figure 6 (triangles) shows that addition of NADHYADH to the phosphate buffer solution produced a significant decrease in the polarization resistance Rp, which is defined as an intersection of an extrapolated EIS curve in a Nyquist plot with the real axis61 (dotted lines):

Rp ) lim [(Zf)real] ω|f0

(7)

At the same time, it was noticed that the capacitance of the double layer (CPEdl) remained constant. Polarization resistance could be used as a measure of the reaction rate and therefore could be (in the first approximation) related to the amount of the protein adsorbed on the surface. Indeed, when the inverse of the polarization resistance (Rp-1) is plotted against the concentration of the protein in the bulk solution, the curve obtained is quite similar to those from the cyclic voltammetry measurements (Figure 2). However, because of the competitive occurrence of several surface processes at the potential at which the EIS measurements were done, +0.6 V (oxidation of the protein, formation of the Pt oxide film, adsorption of phosphate anions), the Rp values have to be corrected for the partial influence of these individual processes. Hence, the resistance due to the formation of oxide film and oxidation (adsorption) of phosphate, Rox, is subtracted from the polarization resistance, Rp, to give the resistance due to the oxidation of the protein, Roxp:

(Roxp)-1 ) (Rp)-1 - (Rox)-1

(8)

Because Rox values are not measurable in a proteincontaining solution (because of the competitive occurrence of the processes described above), they were calculated from Rp values and values of corresponding surface coverage with the protein θpr previously determined from the cyclic voltammetry measurements:

Rox ) Rp(1 - θpr)

(9)

Further, the relation between Roxp and QADS is

(Roxp)-1 ∝ joxp ∝ QADS ∝ Γ

(10)

Adsorption of Yeast Alcohol Dehydrogenase at Pt

Langmuir, Vol. 17, No. 8, 2001 2477

Table 2. Comparison of Gibbs Free Energy Obtained for YADH and NADH-YADH from EIS Measurements ∆GADS/(3 kJ mol-1 temperature/K

YADH

NADH-YADH

299 313 323

-53 -55 -57

-53 -54 -55

Figure 7 shows the dependence of (Roxp)-1 on the concentration of NADH-YADH in the phosphate buffer solution at 313 K. The presented curve is quite similar to Langmuir type isotherms, and if we assume the validity of relation 10, it is possible to calculated the Gibbs free energy of adsorption from the EIS measurements using the Langmuir isotherm:

c (Roxp)-1

)

1 c + p -1 p -1 BADS(Rox ) max (Rox ) max

(11)

As an example, the experimental data from Figure 7 were modeled using the above equation and a plot of c(Roxp) against c (inset in Figure 7) was linear with a correlation coefficient r2 ) 0.9995. By use of the values of the slope and intercept that were determined from the plot, the affinity constant BADS was calculated and then used to obtain the Gibbs free energy of adsorption. Table 2 shows the Gibbs free energies obtained from the EIS measurements for both proteins. The values reported in the table agree well (within 4%) with those obtained from the cyclic voltammetry measurements (Figure 4), which indicates a high reproducibility of the measurements performed on the system investigated. This is particularly important because separate solutions were prepared and measured independently for the two techniques. Conclusions The interfacial behavior of YADH and NADH-YADH at a Pt surface was studied over the temperature range of 273-353 K in a phosphate buffer solution pH 7.0, using

the cyclic voltammetry and electrochemical impedance spectroscopy techniques. The electrode/electrolyte interface and corresponding surface processes were successfully modeled by applying an equivalent-electrical-circuit approach. It was shown that the surface charge density and corresponding polarization resistance, resulting from protein adsorption and its oxidation, respectively, are directly proportional to the amount of adsorbed protein (surface concentration), indicating that adsorption at anodic potentials is accompanied by the transfer of charge, that is, chemisorption through carboxylate groups on the protein. The adsorption isotherms for both proteins showed very high affinity of the proteins toward adsorption onto a Pt surface. The maximum surface concentration values indicate that there is no significant difference in the amount of adsorbed proteins between YADH and NADHYADH in the whole temperature range investigated. The adsorption process was described with the Langmuir adsorption isotherm. From the calculated Gibbs energies of adsorption, it was concluded that both proteins strongly adsorb onto the Pt surface via chemisorption. The adsorption process for YADH was found to be slightly exothermic, whereas the adsorption of NADH-YADH was endothermic, presumably resulting from the excess energetics required for the breaking of intramolecular interactions relative to those involved in the formation of proteinmetal bonds, because of the presence of the coenzyme, NAD+/NADH. The adsorption of both proteins was found to be an entropically governed process, also suggesting structural unfolding of the proteins at the electrode surface. Therefore, the evidence from the present work suggests that disruption of tertiary structure of the proteins occurs upon adsorption at the Pt surface and that the breaking of intramolecular interactions during the adsorption governs the rate of the process. Acknowledgment. Grateful acknowledgment is made to the Natural Science and Engineering Research Council of Canada for support of this research. LA0007729